This invention relates to compounds and methods for enzyme inhibition. In particular, the invention relates to therapeutic methods deriving from enzyme inhibition.
In eukaryotes, protein degradation is predominately mediated through the ubiquitin pathway in which proteins targeted for destruction are ligated to the 76 amino acid polypeptide ubiquitin. Once targeted, ubiquitinated proteins then serve as substrates for the 26S proteasome, a multicatalytic protease, which cleaves proteins into short peptides through the action of its three major proteolytic activities. While having a general function in intracellular protein turnover, proteasome-mediated degradation also plays a key role in many processes such as major histocompatibility complex (MHC) class I presentation, apoptosis, cell division, and NF-xcexaB activation.
The 20S proteasome is a 700 kDa cylindrical-shaped multicatalytic protease complex comprised of 28 subunits organized into four rings. In yeast and other eukaryotes, 7 different xcex1 subunits form the outer rings and 7 different xcex2 subunits comprise the inner rings. The xcex1 subunits serve as binding sites for the 19S (PA700) and 11S (PA28) regulatory complexes, as well as a physical barrier for the inner proteolytic chamber formed by the two xcex2 subunit rings. Thus, in vivo, the proteasome is believed to exist as a 26S particle (xe2x80x9cthe 26S proteasomexe2x80x9d). In vivo experiments have shown that inhibition of the 20S form of the proteasome can be readily correlated to inhibition of 26S proteasome. Cleavage of amino-terminal prosequences of xcex2 subunits during particle formation expose amino-terminal threonine residues, which serve as the catalytic nucleophiles. The subunits responsible for catalytic activity in proteaseome thus possess an amino terminal nucleophilic residue, and these subunits belong to the family of N-terminal nucleophile (Ntn) hydrolases (where the nucleophilic N-terminal residue is, for example, Cys, Ser, Thr, and other nucleophilic moieties). This family includes, for example, penicillin G acylase (PGA), penicillin V acylase (PVA), glutamine PRPP amidotransferase (GAT), and bacterial glycosylasparaginase. In addition to the ubiquitously expressed xcex2 subunits, higher vertebrates also possess three xcex3-interferon-inducible xcex2 subunits (LMP7, LMP2 and MECL1), which replace their normal counterparts, X, Y and Z respectively, thus altering the catalytic activities of the proteasome. Through the use of different peptide substrates, three major proteolytic activities have been defined for the eukaryote 20S proteasome: chymotrypsin-like activity (CT-L), which cleaves after large hydrophobic residues; trypsin-like activity (T-L), which cleaves after basic residues; and peptidylglutamyl peptide hydrolyzing activity (PGPH), which cleaves after acidic residues. Two additional less characterized activities have also been ascribed to the proteasome: BrAAP activity, which cleaves after branched-chain amino acids; and SNAAP activity, which cleaves after small neutral amino acids. The major proteasome proteolytic activities appear to be contributed by different catalytic sites, since inhibitors, point mutations in xcex2 subunits and the exchange of xcex3 interferon-inducing xcex2 subunits alter these activities to various degrees.
The 20S proteasome plays important roles in cell growth regulation, major histocompatibility complex class I presentation, apoptosis, antigen processing, NF-xcexaB activation, and transduction of pro-inflammatory signals.
Small molecules which have been used to inhibit proteasome activity include lactacystin, and short peptides including aldehyde, vinyl sulfone, boronic acid and glyoxal functional groups. These compounds generally lack the specificity, stability, or potency necessary to explore the roles of the proteasome at the cellular and molecular level. For example, peptide aldehydes also inhibit lysosomal and Ca+2-activated proteases, thus complicating a precise dissection of their effects on cells. Vinyl sulfone-based inhibitors have been reported to bind and inhibit intracellular cysteine proteases (for example, cathepsin S), in addition to their actions against the proteasome. Lactacystin has a rate of proteasome inactivation which is significantly slower than that of vinyl sulfone peptide inhibitors. Lactacystin is also non-specific for 20S proteasome, as it has been found to significantly decrease the hydrolysis rate of human platelet lysosomal cathepsin A-like enzyme at pH 5.5.
Enzyme inhibitors are valuable tools that enable the elucidation of details in cellular events that are regulated by these enzymes. Additionally, enzyme inhibitors have therapeutic applications and can be used to carry out mechanistic studies of the machinery of enzymatic processes. The invention relates to the discovery that classes of molecules known as peptide xcex1xe2x80x2,xcex2xe2x80x2-epoxides and peptide xcex1xe2x80x2,xcex2xe2x80x2-aziridines can bind efficiently, irreversibly and selectively to N-terminal nucleophile (Ntn) hydrolases, and can specifically inhibit particular activities of enzymes having multiple catalytic activity.
Once thought merely to dispose of denatured and misfolded proteins, the proteasome is now recognized as constituting proteolytic machinery that regulates the levels of diverse intracellular proteins through their degradation in a signal-dependent manner. Hence, there is great interest in identifying reagents that can specifically perturb the activities of the proteasome and other Ntn hydrolases and thereby be used as probes to study the role of these enzymes in biological processes. Compounds that target the Ntn hydrolases are herein described, synthesized and investigated. Peptide epoxides and peptide aziridines that can potently, selectively, and irreversibly inhibit particular proteasome activities are disclosed and claimed.
Particular peptide epoxides and peptide aziridines modify three catalytic subunits of the 20S proteasome resulting in inhibition primarily of the chymotrypsin-like activity; the trypsin-like and PGPH activities were also inhibited at approximately 100-fold and 1000-fold slower rates, respectively. Furthermore, in comparison with other potent irreversible proteasome inhibitors, peptide epoxides and peptide aziridines inhibit the chymotrypsin-like activity at least about 80-fold faster than lactacystin and at least about four-fold faster than clasto-lactacystin xcex2-lactone. Even higher rates are obtainable.
Other particular peptide epoxides and peptide aziridines primarily inhibit PGPH activity, while having far less inhibitory effect on chymotrypsin-like activity, and virtually no effect on trypsin-like activity. In contrast to the enzyme inhibitors described above, which are highly specific for chymotrypsin-like activity of the proteasome, these other particular PGPH-specific peptide epoxides and peptide aziridines inhibit a catalytic step which is believe to be a rate-limiting step in protein degradation. Their use in elucidating the role(s) of other proteasomal subunits is thus limited. The PGPH-specific inhibitors allow separation of contributions of this particular catalytic activity in biological processes mediated by the proteasome.
Unlike several other peptide-based inhibitors, the peptide epoxides and peptide aziridines described herein do not substantially inhibit non-proteasomal proteases such trypsin, chymonypsin, cathepsin B, papain, and calpain at concentrations up to 50 xcexcM. At higher concentrations, inhibition is observed, but is competitive and not irreversible, since the inhibitor merely competes with the substrate. The novel peptide epoxides and peptide aziridines are also shown to inhibit NF-xcexaB activation and to stabilize p53 levels in cell culture. Moreover, we have demonstrated the potent anti-inflammatory activity of peptide epoxides and peptide aziridines in a mouse model of cutaneous inflammation. Thus, these compounds can be unique molecular probes, which have the versatility to explore Ntn enzyme function in normal biological and pathological processes.
In one aspect, the invention provides N-terminal nucleophile hydrolase inhibitors comprising a heteroatom-containing, three-membered ring, where the ring is bonded to an electron-withdrawing group, and the electron-withdrawing group is bonded to a peptide moiety. These inhibitors can inhibit catalytic activity of N-terminal nucleophile hydrolase enzymes (for example, the 20S proteasome, or the 26S proteasome) when said inhibitor is present at concentrations below about 50 xcexcM, and do not inhibit catalytic activity of non-proteasomal proteases when the inhibitor is present at concentrations below about 50 xcexcM. Regarding the 20S proteasome, particular hydrolase inhibitors inhibit chymotrypsin-like activity of the 20S proteasome when the inhibitor is present at concentrations below about 5 xcexcM, and does not inhibit trypsin-like activity or PGPH activity of the 20S proteasome when is present at concentrations below about 5 xcexcM. Other particular hydrolase inhibitors inhibit PGPH activity of the 20S proteasome when the inhibitor is present at concentrations below about 50 xcexcM, and does not inhibit chymotrypsin-like or trypsin-like activity of the 20S proteasome when the inhibitor is present at concentrations below about 50 xcexcM. The hydrolase inhibitor can be, for example, a peptide xcex1xe2x80x2,xcex2xe2x80x2-epoxy ketone or xcex1xe2x80x2,xcex2xe2x80x2-aziridine ketone, and the peptide can be a tetrapeptide. The tetrapeptide can include branched or unbranched side chains such as hydrogen, C1-6 alkyl, C1-6 hydroxy alkyl, C1-6 alkoxy alkyl, aryl, and aryl-substituted C1-6 alkyl, C1-6 amide, C1-6 amine, C1-6 carboxylic acid, C1-6 carboxyl ester, C1-6 thiol, or C1-6 thioether, for example isobutyl, 1-naphthyl, phenylmethyl, and 2-phenylethyl. The xcex1xe2x80x2-carbon of the xcex1xe2x80x2,xcex2xe2x80x2-epoxy ketone or xcex1xe2x80x2,xcex2xe2x80x2-aziridine ketone can be a chiral carbon atom, such as an (R) or xcex2 configured carbon, as these are defined herein.
In another aspect, the invention includes a method of making a peptide xcex1xe2x80x2,xcex2xe2x80x2-epoxy ketone or xcex1xe2x80x2,xcex2xe2x80x2-aziridine ketone. The method involves synthesizing a first molecule by providing a tripeptide; acetylating the amino terminal of the tripeptide to make an acetylated tripeptide; and catalytically hydrogenating the acetylated tripeptide to make the first molecule. The method further involves synthesizing a second molecule by alkenylating a Weinreb amide of an amino acid having an amino terminal protection group to form an xcex1xe2x80x2,xcex2xe2x80x2-unsaturated ketone; forming a three-membered, heteroatom-containing ring at the xcex1xe2x80x2,xcex2xe2x80x2-unsaturation side to form an xcex1xe2x80x2,xcex2xe2x80x2-epoxy ketone or an xcex1xe2x80x2,xcex2xe2x80x2-aziridine ketone; removing the amino terminal protection group to form the second molecule. The method also involves coupling the first and second molecules to make a peptide xcex1xe2x80x2,xcex2xe2x80x2-epoxy ketone or xcex1xe2x80x2,xcex2xe2x80x2-aziridine ketone. If the tripeptide has hydroxy side chains, protecting the hydroxy side chain to make a protected hydroxy side chain-containing tripeptide; and deprotecting the hydroxy side chain after said coupling are also desirable.
In another aspect, the invention provides pharmaceutical compositions, including a pharmaceutically acceptable carrier, and a pharmaceutically effective amount of the hydrolase inhibitor, which ameliorates the affects of Alzheimer""s disease, muscle-wasting diseases, cancer, chronic infectious diseases, fever, muscle disuse, denervation, nerve injury, and wasting, among others;
In another aspect, the invention provides anti-inflammatory compositions.
In another aspect, the invention provides methods for the following: inhibiting or reducing HIV infection in a subject; affecting the level of viral gene expression in a subject; altering the variety of antigenic peptides produced by the proteasome in an organism; determining whether a cellular, developmental, or physiological process or output in an organism is regulated by the proteolytic activity of a particular Ntn hydrolase; treating Alzheimer""s disease in a subject; reducing the rate of muscle protein degradation in a cell; reducing the rate of intracellular protein degradation in a cell; reducing the rate of p53 protein in a cell; inhibiting the growth of p53-related cancers in a subject; inhibiting antigen presentation in a cell; suppressing the immune system of a subject; inhibiting IxcexaB-xcex1 degradation in an organism; reducing the content of NF-xcexaB in a cell, muscle, organ or subject; affecting cyclin-dependent eukaryotic cell cycles; treating proliferative disease in a subject; affecting proteasome-dependent regulation of oncoproteins in a cell; treating cancer growth in a subject; treating p53-related apoptosis in a subject; and screening proteins processed by N-terminal nucleophile hydrolases in a cell. Each of these methods involves administering or contacting an effective amount of a composition comprising the hydrolase inhibitors disclosed herein, to a subject, a cell, a tissue, an organ or an organism.
In a further aspect, the invention provides a method of making an xcex1,xcex2-aziridine ketone, the method including reacting an xcex1-halo ketone with a) a boron-containing reagent, and with b) an imine for a time and under conditions sufficient to form an xcex1,xcex2-aziridine ketone.
As used herein, the term xe2x80x9cinhibitorxe2x80x9d is meant to describe a compound that blocks or reduces an activity of an enzyme (for example, inhibition of proteolytic cleavage of standard fluorogenic peptide substrates such as suc-LLVY-AMC (SEQ ID NO:1), Box-LLR-AMC and Z-LLE-AMC, inhibition of various catalytic activities of the 20S proteasome). An inhibitor can act with competitive, uncompetitive, or noncompetitive inhibition. An inhibitor can bind reversibly or irreversibly, and therefore the term includes compounds that are suicide substrates of an enzyme. An inhibitor can modify one or more sites on or near the active site of the enzyme, or it can cause a conformational change elsewhere on the enzyme.
As used herein, xe2x80x9cenzymexe2x80x9d can be any partially or wholly proteinaceous molecule which carries out a chemical reaction in a catalytic manner. Such enzymes can be native enzymes, fusion enzymes, proenzymes, apoenzymes, denatured enzymes, farnesylated enzymes, ubiquitinated enzymes, fatty acylated enzymes, gerangeranylated enzymes, GPI-linked enzymes, lipid-linked enzymes, prenylated enzymes, naturally-occurring or artificially-generated mutant enzymes, enzymes with side chain or backbone modifications, enzymes having leader sequences, and enzymes complexed with non-proteinaceous material, such as proteoglycans, proteoliposomes. Enzymes can be made by any means, including natural expression, promoted expression, cloning, various solution-based and solid-based peptide syntheses, and similar methods known to those of skill in the art.
As used herein, the term xe2x80x9cpeptidexe2x80x9d includes not only standard amide linkage with standard xcex1-substituents, but commonly utilized peptidomimics, other modified linkages, non-naturally occurring side chains, and side chain modifications, as detailed below.
As used herein, the term xe2x80x9cheteroatom-containing, three-membered ringxe2x80x9d includes moieties with two carbon atoms and a single heteroatom, such as oxygen or nitrogen.
As used herein, the term xe2x80x9ctreatingxe2x80x9d includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in manner to improve or stabilize a subject""s condition.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.